Thanks to everybody that answered my post about "tilted bands" in my
formaldehyde/MOPS gels.
Here is a compendium of the answers I recieved, (not including those
posted already). One thing I noticed is that I suspect there are as many
ideas for RNA gels as there are people doing them. If anyone has an
really good idea other than those listed below I would love to hear it.
Andrew
______________________________________________________________________________
Andrew Simmonds - Department of Genetics - University of Alberta CANADA
ASIMMOND at GPU.SRV.UALBERTA.CAASIMMOND at VM.UCS.UALBERTA.CA
G216 Biological Sciences Centre
Edmonton, Alberta, CANADA T6G 2E9
******************************************************************************
"Does steel wool come from magnetic sheep?"
******************************************************************************
>>
Date: Sun, 27 Mar 94 09:05:34 EST
From: Carlisle Landel <landel at medsci.udel.edu>
Message-Id: <9403271405.AA07601 at helios.medsci.udel.edu>
To: asimmond at gpu.srv.ualberta.ca
Subject: Re: RNA Gels...
Newsgroups: bionet.molbio.methds-reagnts
In-Reply-To: <2n2ha1$39c at quartz.ucs.ualberta.ca>
Organization: Alfred I. DuPont Institute, Wilmington DE USA
Cc:
Andrew,
I think your problem is caused by a difference in ionic strength
between your gel and your running buffer. Weigh the flask in
which you are melting your agar before and after you boil it, and
you can add back the lost water.
Good luck!
Regards,
Carlisle Landel
>>Date: Sun, 27 Mar 94 19:13:49 -0500
From: coady at ERE.UMontreal.CA (Michael Coady)
Message-Id: <9403280013.AA18767 at alize.ERE.UMontreal.CA>
To: asimmond at gpu.srv.ualberta.ca
Subject: Re: RNA Gels...
Newsgroups: bionet.molbio.methds-reagnts
In-Reply-To: <2n2ha1$39c at quartz.ucs.ualberta.ca>
Organization: Universite de Montreal
Cc:
In article <2n2ha1$39c at quartz.ucs.ualberta.ca> you write:
>I am sure this has been covered before....
>I am having trouble getting TIGHT bands with Northern blots from
>formaldehyde RNA gels... When I cut off the ladder from the gel and look
>at it sideways the bands are "tilted" i.e. ( / ) and I am sure this is a
>problem when I transfer the RNA to a membrane causing the wide bands I am
>seeing. I put 2.2M formaldehyde in the gel and use a standards MOPS
>buffer...
>Might anybody suggest a better way?
>>Andrew Simmonds
Dear Andrew,
There was a brief report published on this in Biotechniques
not too long ago. They found the same thing, and blamed it on a
gradual leaching of the formaldehyde from the gel into the buffer,
resulting in a formaldehyde gradient in the gel the length of the
thickness (if you can follow what I mean). They used a lower
concentration of formaldehyde, and they put it into the buffer as
well as the gel. If you can't find the reference, let me know and
I'll dig it up and send it to you.
Mike
--
Michael J. Coady
COADY at ERE.UMONTREAL.CA
The opinions expressed above are solely those of the author and do not,
in any way, shape or form, represent the Universite de Montreal.
>>From: coady at ERE.UMontreal.CA (Michael Coady)
Message-Id: <9403280026.AA18936 at alize.ERE.UMontreal.CA>
Subject: Re: RNA Gels...
To: asimmond at gpu.srv.ualberta.ca (Andrew Simmonds)
Date: Sun, 27 Mar 1994 19:26:05 -0500 (EST)
In-Reply-To: <Pine.3.87.9403271700.A4698-0100000 at gpu.srv.ualberta.ca> from "Andrew Simmonds" at Mar 27, 94 05:25:00 pm
X-Mailer: ELM [version 2.4 PL21]
Mime-Version: 1.0
Content-Type: text/plain; charset=US-ASCII
Content-Transfer-Encoding: 7bit
Content-Length: 456
>> Thank you for the advice...
> I will look for the reference... but can I also assume that if the
> gradient would correspond to the thickness of the gel, then perhaps a
> thinner gel would help?
> Andrew
>>
Well, I suppose it would, but it's awfully easy to just add
formaldehyde to the buffer, particularly since they are using
something like 0.6 M formaldehyde rather than 2.2 as you're
using. I'll see if I can dig it up right now.
Mike
>>Date: Mon, 28 Mar 1994 15:13:07 -0400 (EDT)
From: Liam E Good <lgood at uoguelph.ca>
Subject: Re: RNA Gels...
To: Andrew Simmonds <asimmond at gpu.srv.ualberta.ca>
In-Reply-To: <2n2ha1$39c at quartz.ucs.ualberta.ca>
Message-Id: <Pine.3.07.9403281502.A2207-b100000 at herman.cs.uoguelph.ca>
Mime-Version: 1.0
Content-Type: TEXT/PLAIN; charset=US-ASCII
On 26 Mar 1994, Andrew Simmonds wrote:
> I am sure this has been covered before....
> I am having trouble getting TIGHT bands with Northern blots from
> formaldehyde RNA gels... When I cut off the ladder from the gel and look
> at it sideways the bands are "tilted" i.e. ( / ) and I am sure this is a
> problem when I transfer the RNA to a membrane causing the wide bands I am
> seeing. I put 2.2M formaldehyde in the gel and use a standards MOPS
> buffer...
> Might anybody suggest a better way?
Hi Andrew,
I don't have the reference at hand, but someone figured that the tilting
was due to the formaldehyde diffusing out of the gel during the run so
that the gel ends up having a gradient of formaldehyde from bottom to top
which presumably causes the slanting of the bands. It is pretty nasty to
equalize the formaldehyde conc. by making your running buffer 2.2M
formaldehyde. I think the best solution (suggested in some Biotechniques
note some time ago) is to reduce the formaldehyde conc. about 10 fold.
That way you get lest of a gradient forming. Apparently it is still
enough formaldehyde to prevent the RNA from forming secondary structures.
Also, the gel does not smell so bad and it won't break as easily. It has
been working for me. Give it a try.
Let me know if it straightens-out your problem.
Good Luck,
Liam
>>Date: Mon, 28 Mar 1994 21:00:48 -0400
From: RICHARD ISBRUCKER <ISBRUCK at ac.dal.ca>
Subject: Re: RNA Gels...
To: asimmond at gpu.srv.ualberta.ca
Message-Id: <01HAIPH7VD0I008TVI at AC.DAL.CA>
X-Vms-To: dal1::in%"asimmond at gpu.srv.ualberta.ca"
X-Vms-Cc: ISBRUCK
Mime-Version: 1.0
Content-Type: TEXT/PLAIN; CHARSET=US-ASCII
Content-Transfer-Encoding: 7BIT
>From: asimmond at gpu.srv.ualberta.ca (Andrew Simmonds)
>Subject:RNA Gels...
>Date: 26 Mar 1994 23:42:57 GMT
>I am sure this has been covered before....
>I am having trouble getting TIGHT bands with Northern blots from
>formaldehyde RNA gels... When I cut off the ladder from the gel and look
>at it sideways the bands are "tilted" i.e. ( / ) and I am sure this is a
>problem when I transfer the RNA to a membrane causing the wide bands I am
>seeing. I put 2.2M formaldehyde in the gel and use a standards MOPS
>buffer...
>Might anybody suggest a better way?
>>Andrew Simmonds
Hello Andrew,
I noticed the same thing happening to my RNA gels two years ago. I wasn't
too sure what caused it or why it happened, but several people offered me
different pieces of advice. Since I was in a rush at the time, I combined
most of the advice together at once and the problem went away... therefore,
I am not too sure which one(s) solved the problem. Here's what I did to
correct the problem:
1- Use new agarose and don't use any that is greater than 1 year old.
2- Circulate the mops buffer using a peristaltic pump in order to prevent
differences in pH between the +'ve and -'ve sides of the aparatus.
3- Keep the agarose hot (ie >90C) for at least 5 minutes to ensure it has
all dissolved. Add the formaldehyde, MOPS, etc., only when the agarose
is below 75C and add these slowly with stirring.
4- Use 0.66M formaldehyde instead of 2.2M
5- Poor the hot agarose rapidly and spread it by gentle swirling. It should
still be very liquid when it is poored... agarose that has started to set
when it is poored does not have equal thickness or density throughout.
6- Poor the agarose in a warm location and do not cover it (to prevent
condensation). I put my gel former on a metal plate that was pre-warmed
to 37C.
7- Use a very low voltage during electrophoresis. I now do mine for 18 hours
at 5 volts. (In a 1% agarose gel the rRNA bands migrate 4.5 and 6.5 cm.)
8- Keep the gels thin (ie 3 mm or less).
Hope these tips help. Good luck,
Richard Isbrucker
--
Richard Isbrucker ISBRUCK at ac.dal.ca
Dalhousie University (902) 494-2571
Department of Pharmacology
Halifax, Nova Scotia, Canada
B3H 4H7
--
>>Date: Thu, 31 Mar 1994 16:32:39 +1200 (NZST)
From: MKENNEDY at CHMEDS.AC.NZ
To: asimmond at gpu.srv.ualberta.ca
Message-Id: <940331163239.76a8 at CHMEDS.AC.NZ>
Subject: Re: RNA Gels...
Date sent: 31-MAR-1994 16:21:22
Hi Andrew
>Might you suggest a good reference for a novice to glyoxyl gels?
>I am at the stage where I am taking a rest from Northern's and collecting
>info for the re-grouping and second attack...
Here is the protocol I use; I'm afraid the format and all Greek "mu" and "
alpha" characters, and a few others, get converted to * when i shift stuff off
my PC onto the mainframe, but I think it should be clear enough.
Cheers,
Martin
NNNN NN Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz) ZZZZZZZ
NN NN NN Cytogenetic and Molecular Oncology Unit ZZZ
NN NN NN Christchurch School of Medicine ZZZ
NN NNNN Christchurch, New Zealand ZZZZZZZ
Phone (64-3)364-0880 Fax (64-3)364-0750
Northern Blots
RNA can be completely denatured by glyoxalating the bases in the presence of
DMSO, thus prohibiting base pairing. RNAs denatured in this way will migrate
in agarose or PAGE gels without interference because of secondary structure.
However, samples treated in this way should be relatively 'clean', so that the
glyoxal is not depleted by contaminanting junk.
Care should be taken with the equipm ent and reagents used in this procedure.
A gel tank and combs should be set aside exclusively for use with Northerns.
If in doubt, scrub down gear with 10% SDS, 0.5M NaOH and rinse well with DI
water. I keep a separate sponge in a sealed plastic box for Northern blots
only. Beware of sponges that have seen NaOH! Do not recycle the buffer; the
sodium phosphate buffer breaks down and should not be kept for longer than a
week.
References:
McMaster and Carmichael 1977 PNAS USA 74:4835
Maniatis, et al 1982 "Molecular Cloning" CSH
Davis, et al 1980 "Advanced Bacterial Genetics" CSH pp156-158
A. Sample preparation:
1. Mix the following in a microfuge tube:
60 *l glyoxal
20 *l 0.2M sodium phosphate, pH 7.0
Dispense 4 *l aliquots into required number of tubes. Discard remaining mix.
2. To each tube, add 10 *g of RNA and MPW as necessary to give a final
volume of 18 *l . If RNA is too dilute then concentrate by precipitation
before use.
3. Incubate capped tube at 50 oC for 1 hr. Chill on ice and spin briefly.
Add 4 *l loading dye.
4. Run on 1.4% agarose gel made up with 10 mM sodium phosphate buffer,
pH7.0, not TBE. Electrophorese in the same buffer, at 100mA. Make sure the
buffer covers the gel by a few mm, and place the gel tank on two magnetic
stirrers, with fleas in each resrvoir. Begin stirring once the loading dye has
entered the gel, and take care that the gel doesn't float free during the run -
two glass pipettes can be laid down the sides of the gel to keep it in place
if necessary. Stirring must be fairly brisk, as the pH can change rapidly and
the glyoxal will dissociate from the RNA. Run until the bromophenol blue is
about > way down the gel.
B. Blotting:
1. Transfer directly to Hybond-N+ in 20xSSC with no pre-treatment. Wet
the membrane in DI water, then soak in 20x SSC for about 15 minutes. Set up
blot as usual, except that 20x SSC is used for the transfer.
2. After transfer (usually overnight) mark the position of slots on the
membrane, using a Pilot pen. Do not wash filter, or soak in SSC. Look at
filter under hand-held UV lamp, and mark the 28s and 18s bands with arrows on
both edges of filter.
3. UV-crosslink RNA to membrane while still damp (auto-crosslink in
Stratalinker) and then bake at 80 oC for one hour (to de-glyoxylate the RNA).
4. Hybridize and wash as usual; it is not necessary to take extraordinary
precautions to prevent RNA degradation at this stage.
C: Stripping:
1. Wet filter in 6x SSC, 0.1% SDS.
2. Wash at 65 oC in 50% formamide, 10mM sodium phosphate pH 7.0, for one
hour.
3. Wash 2x 15 minutes in 6x SSC, 0.1% SDS at 65 oC. Store at -20 oC.
Solutions:
Glyoxal: Use Sigma G-3140, 40% aqueous solution. A precipitate may form on
storage, which can be dissolved by heating to 50-60 oC. De-ionize 10ml with
5g mixed bed resin (TMD-8). Do this in a yellow-top tube, on a roller or
rocker for an hour or so. Store 100 *l aliquots at -20 oC.
Sodium phosphate buffer: Combine 6.1g NaH2PO4 2H20 and 8.65g Na2HPO4 (
anhydrous) in 500ml DI water. pH should be 7.0 (check by adding a drop onto
some pH paper). Make fresh each time. Keep a bottle set aside purely for
this use.
Loading dye: 50% glycerol; 10mM sodium phosphate buffer, pH7.0; 0.25%
bromophenol blue. Store 100 *l aliquots at -20 oC.
>>
To: asimmond at gpu.srv.ualberta.ca (Andrew Simmonds)
Subject: Re: RNA Gels...
Newsgroups: bionet.molbio.methds-reagnts
Organization: Institut National de la Recherche Agronomique - JOUY
Andrew,
Have a look to Biotechniques, 14 (3) 380, 1993. The authors investigated the
reasons for the broadness of the ribs' RNA bands and found that, when the
gels were viewed from the side, the bands were tilted. Their conclusion is
that tilting is associated with a formaldehyde gradient in the gel, and they
recommend to add formaldehyde to the buffer in the same concentration as in
the gel. In order to minimize exposure to formaldehyde, they also recommend
to use 0.22 M formaldehyde.
For the same reason, broad ribs'RNA bands, I'm using methylmercury gels,
using a chemical hood that I'll use also with formaldehyde...
The point is that, in order to prevent methylmercury from diffusing out of
the gel, we cover the gel with Parafilm (I'm no t connected with the company...)
or with a block of perpex. As I'm thinking of switching from methylmercury to
formaldehyde, I've tried the same trick with formaldehyde gels, and, although I do not have a large experience with this new system, it seems to work, at least when looking to the ethidium bromide colored RNAs. So, if my experience is
valid, just cover your gel !
Concerning methylmercury gels, they really give very nice thin bands. In order
to reduce the amount of toxic used, we add methylmercury in the sample, but
not in the gel. This works perfectly, at least until now.
Hope this help !
Francois Hatey
Laboratoire de Genetique Cellulaire
Centre INRA de Toulouse
BP 27
31326 Castanet Tolosan Cedex
France
FrancoisHatey at toulouse.inra.fr